Secretory granules (Sgs) of endocrine cells are responsible for storage and regulated secretion of peptide hormones including insulin. Despite recent progress, the mechanisms responsible for SG biogenesis and exocytosis remain unclear. We have previously shown that islet cell autoanigen (ICA) 512, a new autoantigen of autoimmune diabetes with homology to receptor protein tyrosin phosphatase (RPTPase), is an intrinsic membrane protein of Sgs. We hypothesize that ICA 512, and thus tyrosine phophorylation/dephosphorylation, is in volved in regulating SG trafficking, including their biogenesis or exocytosis. We will test this hypothesis by measuring whether SG biogenesis or exocytosis is affected in rat insulinoma (RIN) m5F cells stably transfected with ICA512 mutants. As an alternative approach to investigate the role of ICA 512 in SG biogenesis we will apply a cell-free assay in which SG formation is reconstituted in vitro . SG biogeneiss in vitro will be measured upon addition of protein tyrosine kinase (PTK) and PTPase inhibitors as well as recombinant ICA 512 or antiICA512. We will also determine whether the intravesicular domain of ICA 512 binds to the granins, a family of regulated secretory proteins which may play a role in SG biogenesis. As an additional approach to investigate ICA 512's participation in SG exocytosis, rat pheochromocytoma PC12 cells will be transiently co-transfected with ICA 512 mutants and green fluorescent protein (GFP) as a reporter gene. Living single co-transfected PC 12 cells will be identified by microscopy as GFP-positive. Dopamine release from Sgs of these cells will be measured using carbon fibers as electrochemical detectors. Pro-ICA 512 cleavage generates two fragments: a transmembrane fragment which remains associated with Sgs and a luminal fragment the fate of which is unknown. Our hypothesis we will search for ICA 512 derived fragments in the culture media of RIN m5F cells and in human sera. If found, we will use pulse-chase experiments in RIN m5F cells, we will determine whether these ICA 512 derived peptides are secreted in a regulated or constitutive fashion and in which compartment pro-ICA 512 is cleaved. We also aim to identify the protein convertase that cleaves pro-ICA 512 by co-transfecting ICA 512 with protein convertases into COS cells, a cell line that does not endogenously express ICA 512 and it is unable to process it upon its transfection.